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Regulatory T cells (Tregs) exert inhibitory function under various physiological conditions and adopt diverse characteristics following environmental cues. Multiple subsets of Tregs expressing master transcription factors of helper T cells such as RORγt, T‐bet, Gata3 and PPARγ have been characterized, but the molecular mechanism governing the differentiation of these subsets remains largely unknown. Here we report that the atypical IκB protein family member Bcl‐3 suppresses RORγt+ Treg accumulation. The suppressive effect of Bcl‐3 was particularly evident in the mouse immune tolerance model of anti‐CD3 therapy. Using conditional knockout mice, we illustrate that loss of Bcl‐3 specifically in Tregs was sufficient to boost RORγt+ Treg formation and resistance of mice to dextran sulfate sodium‐induced colitis. We further demonstrate the suppressive effect of Bcl‐3 on RORγt+ Treg differentiation in vitro. Our results reveal a novel role of nuclear factor‐kappa B signaling pathways in Treg subset differentiation that may have clinical implications in immunotherapy. [ABSTRACT FROM AUTHOR]

Bcl-3 is an atypical member of the IκB family that acts in the nucleus to modulate transcription of many NF-κB targets in a highly context-dependent manner. Accordingly, complete Bcl-3-/- mice have diverse defects in both innate and adaptive immune responses; however, direct effects of Bcl-3 action in individual immune cell types have not been clearly defined. Here, we document a cell-autonomous role for Bcl-3 in CD8+ T cell differentiation during the response to lymphocytic choriomeningitis virus infection. Single-cell RNA-seq and flow cytometric analysis of virus-specific Bcl3-/- CD8+ T cells revealed that differentiation was skewed towards terminal effector cells at the expense of memory precursor effector cells (MPECs). Accordingly, Bcl3-/- CD8+ T cells exhibited reduced memory cell formation and a defective recall response. Conversely, Bcl-3-overexpression in transgenic CD8+ T cells enhanced MPEC formation but reduced effector cell differentiation. Together, our results establish Bcl-3 as an autonomous determinant of memory/terminal effector cell balance during CD8+ T cell differentiation in response to acute viral infection. Our results provide proof-of-principle for targeting Bcl-3 pharmacologically to optimize adaptive immune responses to infectious agents, cancer cells, vaccines and other stimuli that induce CD8+ T cell differentiation. Author summary: Effective immunity to previously encountered pathogens or vaccines depends on efficient formation of memory T and B cells. A major cornerstone is the generation of memory CD8+ T cells, which kill cells infected with intracellular pathogens, including viruses. In addition, memory CD8+ T cells are critical for adoptive immunotherapy to eliminate targeted tumor cells. Hence, it is essential to have a detailed understanding of pathways and molecular factors that regulate T cell differentiation/memory formation during the initial immune effector response. Here we show a critical cell-autonomous role for Bcl-3, an atypical member of the IκB family, in the regulation of CD8+ T cell differentiation/memory formation during acute viral infection with lymphocytic choriomeningitis virus. We found that Bcl-3 dampens the terminal differentiation of CD8+ T cells, which normally results in death at the end of the primary effector response, and instead, promotes the formation of memory precursor cells early on. This led to a robust memory response and much improved viral clearance upon re-challenge. Our results provide new insight into the molecular regulation of CD8+ T cell effector response and memory cell generation, establishing Bcl-3 and its pathways as a target to optimize CD8+ T cell effector responses to various immunogens including infectious agents, cancer cells, and vaccines. [ABSTRACT FROM AUTHOR]

Bcl‐3 is an atypical member of the IκB family that modulates NF‐κB activity in nuclei. lpr mice carry the lpr mutation in Fas, resulting in functional loss of this death receptor; they serve as models for lupus erythematosus and autoimmune lymphoproliferation syndrome (ALPS). To explore the biologic roles of Bcl‐3 in this disease model, we generated BL6/lpr mice lacking Bcl‐3. Unlike lpr mice on an MRL background, BL6/lpr mice present with very mild lupus‐ or ALPS‐like phenotypes. Bcl‐3 KO BL6/lpr mice, however, developed severe splenomegaly, dramatically increased numbers of double negative T cells — a hallmark of human lupus, ALPS, and MRL/lpr mice — and exhibited inflammation in multiple organs, despite low levels of autoantibodies, similar to those in BL6/lpr mice. Loss of Bcl‐3 specifically in T cells exacerbated select lupus‐like phenotypes, specifically organ infiltration. Mechanistically, elevated levels of Tnfα in Bcl‐3 KO BL6/lpr mice may promote lupus‐like phenotypes, since loss of Tnfα in these mice reversed the pathology due to loss of Bcl‐3. Contrary to the inhibitory functions of Bcl‐3 revealed here, this regulator has also been shown to promote inflammation in different settings. Our findings highlight the profound, yet highly context‐dependent roles of Bcl‐3 in the development of inflammation‐associated pathology. [ABSTRACT FROM AUTHOR]

The atypical IκB family member Bcl-3 associates with p50/NF-κB1 or p52/NF-κB2 homodimers in nuclei, thereby either positively or negatively modulating transcription in a context-dependent manner. Previously we reported that Bcl-3 was critical for host resistance to Toxoplasma gondii. Bcl-3-deficient mice succumbed within 3-5 weeks after infection, correlating with an apparently impaired Th1-type adaptive immune response. However in which cell type(s) Bcl-3 functioned to assure resistance remained unknown. We now show that Bcl-3 expression in dendritic cells is required to generate a protective Th1-type immune response and confer resistance to T. gondii. Surprisingly, mice lacking Bcl-3 in dendritic cells were as susceptible as mice globally deficient for Bcl-3. Furthermore, early innate defenses were not compromised by the absence of Bcl-3, as initial production of IL-12 by dendritic cells and IFN-γ by NK cells were preserved. However, subsequent production of IFN-γ by CD4+ and CD8+ T-cells was compromised when dendritic cells lacked Bcl-3, and these mice succumbed at a time when T-cell-mediated IFN-γ production was essential for host resistance. These findings demonstrate that Bcl-3 is required in dendritic cells to prime protective T-cell-mediated immunity to T. gondii. [ABSTRACT FROM AUTHOR]

Regulatory T cells (Tregs) exert inhibitory function under various physiological conditions and adopt diverse characteristics following environmental cues. Multiple subsets of Tregs expressing master transcription factors of helper T cells such as RORγt, T-bet, Gata3 and PPARγ have been characterized, but the molecular mechanism governing the differentiation of these subsets remains largely unknown. Here we report that the atypical IκB protein family member Bcl-3 suppresses RORγt + Treg accumulation. The suppressive effect of Bcl-3 was particularly evident in the mouse immune tolerance model of anti-CD3 therapy. Using conditional knockout mice, we illustrate that loss of Bcl-3 specifically in Tregs was sufficient to boost RORγt + Treg formation and resistance of mice to dextran sulfate sodium-induced colitis. We further demonstrate the suppressive effect of Bcl-3 on RORγt + Treg differentiation in vitro. Our results reveal a novel role of nuclear factor-kappa B signaling pathways in Treg subset differentiation that may have clinical implications in immunotherapy., (Published 2021. This article is a U.S. Government work and is in the public domain in the USA.)

Quantification of gene expression and characterization of gene transcript structures are central problems in molecular biology. RNA sequencing (RNA-Seq) and chromatin immunoprecipitation sequencing (anchor-Seq) are important methods, but can be cumbersome and difficult for beginners to learn. To teach interested students and scientists how to analyze RNA-Seq and anchor-Seq data, we present a start-to-finish tutorial for analyzing RNA-Seq and anchor-Seq data: SeqAcademy ( source code:  https://github.com/NCBI-Hackathons/seqacademy, webpage: http://www.seqacademy.org/). This user-friendly pipeline, fully written in markdown language, emphasizes the use of publicly available RNA-Seq and anchor-Seq data and strings together popular tools that bridge that gap between raw sequencing reads and biological insight. We demonstrate practical and conceptual considerations for various RNA-Seq and anchor-Seq analysis steps with a biological use case - a previously published yeast experiment. This work complements existing sophisticated RNA-Seq and anchor-Seq pipelines designed for advanced users by gently introducing the critical components of RNA-Seq and anchor-Seq analysis to the novice bioinformatician. In conclusion, this well-documented pipeline will introduce state-of-the-art RNA-Seq and anchor-Seq analysis tools to beginning bioinformaticians and help facilitate the analysis of the burgeoning amounts of public RNA-Seq and anchor-Seq data., Competing Interests: No competing interests were disclosed., (Copyright: © 2019 Ather SH et al.)

Interleukin-17 (IL-17) is essential in host defense against extracellular bacteria and fungi, especially at mucosal sites, but it also contributes significantly to inflammatory and autoimmune disease pathologies. Binding of IL-17 to its receptor leads to recruitment of adaptor protein CIKS/Act1 via heterotypic association of their respective SEFIR domains and activation of transcription factor NF-κB; it is not known whether CIKS and/or NF-κB are required for all gene induction events. Here we report that CIKS is essential for all IL-17-induced immediate-early genes in primary mouse embryo fibroblasts, whereas NF-κB is profoundly involved. We also identify a novel subdomain in the N terminus of CIKS that is essential for IL-17-mediated NF-κB activation. This domain is both necessary and sufficient for interaction between CIKS and TRAF6, an adaptor required for NF-κB activation. The ability of decoy peptides to block this interaction may provide a new therapeutic strategy for intervention in IL-17-driven autoimmune and inflammatory diseases.

In the present article, we report that DR4 or DR5 overexpression dramatically activates the release of the inflammatory cytokines IL-8, TNF-alpha, CCL20, MIP-2 and MIP-1beta in an NF-kappaB-dependent manner in 293T, MDA-MB-231 and HCT-116 cells. We showed that death receptor-mediated signals were extracellular domain-independent, whereas the effect of overexpression of the DR4 intracellular domain was much less potent. The TRADD-TRAF2-NIK-IKKalpha/beta signaling cascade, which plays an essential role in TNF-induced NF-kappaB activation, was found to be involved in tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor-mediated signal transduction. The FADD-caspase signaling pathway, which has been reported to be mostly related to apoptosis, was identified as being essential for DR4 or DR5 overexpression-mediated NF-kappaB activation and cytokine secretion and crosstalks with the TRADD-TRAF2-NIK-IKKalpha/beta signaling cascade. Furthermore, a DR5 agonistic antibody (AD5-10) triggered the inflammatory cytokine release. These data, together with previous reports, provide strong evidence that TRAIL and TRAIL receptors play an important role in inflammation.